Abstract

The goal of this project was the characterisation of a new guanine nucleotide exchange factor (GEF protein) for Rac GTPases in Dictyostelium discoideum. GEF proteins function as stimulating proteins of Rac GTPases which are of central importance in the regulation of actin-involving processes. Actin is a major component of the cytoskeleton, constituting a dynamic network of filamentous structures which provide the basis for elementary tasks of growth, differentiation, cell movement and cell division.
The ameba D. discoideum is a haploid unicellular eukaryote. It serves as a model organism to study basic actin-involving processes in higher organisms. The D. discoideum genome has been fully sequenced recently and comprises a wide range of regulatory components for the actin cytoskeleton. The gene of a novel GEF protein with a coding sequence of 3597 bp was studied in detail. The gene was cloned and the sequence verified. The protein sequence of 1198 amino acids comprises three Calponin homology domains (CH) and one Dbl homology/Pleckstrin homology tandem domain which is a typical sequence feature of GEF proteins. On the basis of this domain architecture the protein was named 'Trix' in short for 'triple Calponin exchange factor'.
The CH domains were classified as type 1 and type 1 CH domain (type 3 - type 3 - type 1). This resembles a novel combination of CH domains in a Rho GEF protein. A recombinant fragment carrying the three CH domains was shown to bind and bundle actin filaments which is not explained by the functions that have been described for CH domains so far.
In vivo Trix is localised in the area of the actin-rich cell cortex as well as on the membranes of late endocytic vesicles. This suggests a regulatory role for Trix in the assembly or the disassembly of the actin coat that is associated with endocytic vesicles during the late stages of endocytosis. It was shown that Trix is mainly expressed during the vegetative stadium of D. discoideum which would be consistent with the increased endocytosis during growth. An association of Rac GTPases with late endocytic vesicles in D. discoideum was not described so far.
Trix displayed no GDP/MANT-GDP exchange activity with the Rac GTPases Rac1a, RacC and RacE, hence the protein could not be allocated to a Rac GTPase signaling pathway. Some of the interactions between exchange factors and their respective Rac GTPases are of a very specific nature. Thus a potential activity of Trix might be directed against a Rac GTPase that has not yet been tested. The activation of Trix might also depend on further uncharacterised regulatory components. Finally, the results of the in vitro assays might differ from the in vivo situation as it has been demonstrated for other Rho GEFs.
The Trix gene was disrupted in AX2 wildtype cells by a gene replacement approach. This allowed detailed characterisation of the protein's function in vivo. Trix is not an essential protein. There were no significant differences in the expression of important marker proteins, in phototaxis, chemotaxis, phagocytosis, cytokinesis, and growth of Trix- mutants. The mutant cells showed subtle changes in the organisation of the actin system as well as a slight delay during the developmental cycle. The most severe phenotypic deviation displayed by Trix- mutants consisted in a marked reduction of exocytosis. This provides further evidence for a regulatory function of the protein in exocytosis.
The data suggest that Trix plays a role in the organisation of actin-involving processes in D. discoideum, especially in the regulation of the late endocytic cycle. Trix could not be allocated to a specific Rac GTPase signaling pathway and definite structural or dynamic tasks on the basis of the protein’s actin-binding and actin-bundling properties. The subtle phenotypic alterations in Trix- mutants might be due to a general redundancy in the functions of D. discoideum Rho GEF proteins.